Lecture 15. Mammalian Cell Engineering 2

Question 1

What is CD19 (surface antigen)?

Answer 1

CD19 is a classical B cell marker and is expressed on 90% of B cells malignancies (blood cancers); most CAR T cell approaches used in ALL, CLL & similar are target CD19

Question 2

How many CAR T-cell therapies are approved in the UK?

Answer 2

3 out of 6

Question 3

What are the problems with the CAR-T approach

Answer 3

Cytokine release syndrome (‘cytokine storm’)
Suppressive tumour microenvironment
T cell exhaustion (Loses affinity to killing target cells or releasing cytokines)
Lymphopaenia (reduced number of lymphocytes)
Generally not very effective for solid tumours

Question 4

How to overcome immunosuppression

Answer 4

The tumor microenvironments often are immune suppressive and contain many inhibitory factors such as the TGFβ and IL-4 cytokines, or co-inhibitory surface molecules such as PD-L1

Question 5

What ‘Kill switch’ strategies are there for CAR T cells?

Answer 5

CAR T cells are made to express HSV thymidine kinase so add Ganciclovir which causes replication defects
CAR T cells contain inducible Caspase 9 system so induce Caspase 9 expression with small molecule,
which causes apoptosis
CAR T cells are made to express surface receptors or epitope tags so add antibody which recognises surface tag and activates immune response against cells

Question 6

What does TALEN/Cas9 do?

Answer 6

Disrupt endogenous TCR ⇒ can use unmatched donor cells

Question 7

What is SUPRA and what does it do?

Answer 7

split, universal, and programmable Based on ‘leucine zipper’ interaction.

Question 8

What is SynNotch?

Answer 8

Synthetic notch receptor activates expression of a second receptor for a different antigen ‘AND’ logic gate.

Question 9

What is optogenetics?

Answer 9

Combination of genetics and light-mediated control
Minimal ‘invasive’
Precise (cell type specific, e.g. in combination with Cre/Lox)
Fast (millisecond response times)
Popular in neurobiology (manipulation of individual neurons)

Question 10

What happens in light based fear?

Answer 10

Fear conditioning (electric shock to foot) labels cells of the Dentate Gyrus with EYFP and ChR2, memory of fear can be recalled with light
Cells that are responding to fear become labelled yellow due to expression of EYFP

Question 11

What happens in encapsulation?

Answer 11

Encapsulation (e.g. in alginate or cellulose) of engineered cells ensures exchange (i.e. protein or small molecule diffusion) with host but avoids immune reactivity and DNA modification of host

Question 12

What happens with β-cell mimetic designer cells?

Answer 12

1. Engineer cell line with Ca2+ circuit to secrete insulin based on glucose level
2. Add food-related control
   Cell was engineered to excrete insulin, partially exploit existing white type signalling

Question 13

What happens with non-invasive expression system involving radiowaves?

Answer 13

1. Engineer cell line with Ca2+ circuit to secrete insulin; Ca2+ enters upon radiowave-heating of nanoparticles attached to temperature-sensitive channel (TRPV1)
2. Engineer cells to synthesise nanoparticles intracellularly (Ferritin binds to iron and can become responsive to radio waves)

Question 14

What happens in multicellular systems?

Answer 14

The increasing complexity of synthetic genetic circuits poses limits in terms of what is achievable in single cells, mainly due to noise, orthogonality, construction difficulties and similar issues

Question 15

What happens in 3rd part parasitiam?

Answer 15

Yeast cell recognised by HEK cells, start producing ampicillin which helps cell to grow